Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for many malignant diseases but its clinical utility has been impeded by graft-versus-host disease (GVHD). Unfortunately, there are currently no therapies that target proteins that are elevated in the blood of patients presenting with GVHD, and no effort has been made to develop drugs specific for GVHD. This represents an important problem, because currently therapies are limited to the nonspecific targeting of effector cells. Our long-term goal is to develop drugs targeting proteins that are dysregulated in patients with GVHD as potential novel GVHD therapies. Our objective in this application is to discover chemical probes that can inhibit these elevated proteins through high throughput screening (HTS) of small molecules libraries and be evaluated in the biological model systems. These compounds will allow for discovery of potential GVHD-specific drugs and therefore better harnessing of treatment in many patients with hematological cancers. Our central hypothesis is that the lead plasma biomarker of GVHD, suppression of tumorigenicity 2 (ST2) can be targeted with small molecule compounds to alleviate GVHD. This hypothesis was formed based on our published data characterizing the soluble form of ST2 (sST2) as the most significant biomarker to predict the non-response to GVHD therapy. sST2 acts as a decoy receptor for IL-33, the only known ligand for ST2, driving Th2 cells toward a Th1 phenotype. This phenotypic change leads to GVHD, and an inhibitor of the binding association between sST2 and IL-33 could alleviate GVHD. We therefore embark on a pilot HTS campaign using the AlphaScreen detection assay to discover chemical probes that inhibit the ST2 and IL33 interaction. Our optimized assay condition was robust giving z'-factors e 0.7 and permitted discovery of 17 hits. The rationale for this study is that the likelihood for better probes than those in the pilot screen warrants a large screen. In addition, a platform including in silico enrichment of potential hits from broader compound libraries and testing of the chemical probes in biological models of GVHD will be relevant. The three specific aims are: 1) HTS implementation to discover chemical probes that inhibit the ST2/IL33 pathway that is overexpressed in graft-versus-host disease. 2) Enrich hits using focus compound libraries generated from pharmacophore models based on confirmed hits from HTS and identification of putative binding sites in ST2. 3) Hits validation with in vitro huma assays and in vivo murine models of GVHD. This integrative approach is innovative because it applies HTS technology of 176,000 diverse small molecules with in silico enrichment of hits and target binding site evaluation yielding the identification and development of chemical probes against biomarkers of GVHD refractoriness to therapy. The proposed research is significant because discovery of GVHD-specific inhibitors will provide the foundation for therapeutic evaluation of GVHD biomarkers as druggable targets. The application of these drugs will drive the phenotypic changes in appropriate effector T cells with increased efficacy and lowered toxicity.